This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have identified certain regions of the Caulobacter crescentus chromosome that are dynamically localized versus the cell cycle (e.g., the origin and terminus of DNA replication). High-resolution information on positioning of these regions versus cell cycle time (how near the cell poles, the mid-plane, and whether membrane associated) is of high interest. In addition, we have identified numerous dynamically localized regulatory and structural proteins in Caulobacter. Our localization studies have been done by time-lapse fluorescent microscopy following the changing position of GFP-tagged proteins as a function of time in Caulobacter cell cycle. EM tomography will provide higher resolution information on specific positioning of membrane-localized proteins versus various cell surface structures (stalk, flagella and pili base structures and cell division plane). The EM tomography results will provide key insights into the role of dynamic 3D positioning of regulatory proteins in progression of the bacterial cell cycle and into the mechanism of asymmetric cell division.